Methods for increasing blood plasma 2′-deoxyuridine (dUrd) and thymidylate synthase inhibition

ABSTRACT

The present invention provides methods for increasing plasma dUrd levels comprising the administration of 6R-MTHF. The methods of increasing plasma dUrd increase dUrd levels compared to equimolar concentrations of LV. The present invention also provides methods for increasing TS inhibition comprising the administration of 6R-MTHF. The present invention also provides methods for increasing TS inhibition comprising the administration of 6R-MTHF.

The instant application claims priority to U.S. Provisional ApplicationSer. No. 62/458,868 filed Feb. 14, 2017, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the treatment of solid tumors in humanssuch as cancer, which involves the administration of [6R]-5,10-methylenetetrahydrofolate (6R-MTHF) in 5-fluorouracil (5-FU) based chemotherapy.

BACKGROUND OF THE INVENTION

5-Fluorouracil (5-FU) was first introduced in 1957 and remains amainstay in treatment of solid tumors including colorectal cancer (CRC).5-FU exerts cytotoxic activity primarily through inhibition ofthymidylate synthase (TS) and to some extent also through incorporationof metabolites into RNA. (Ford et al. (2002) Clinical Cancer Research2002; 8(1): 103-109). The overall response rate of 5-FU alone is quitelimited (10-15%) and modulation strategies have been developed toincrease the anticancer activity of 5-FU. (Johnston et al. AnticancerDrugs 2001, 12: 639-646). One of the most widely used strategiesinvolves the co-administration of the folate 5-formyl tetrahydrofolate(folinic acid or leukovorin or LV) with 5-FU. (Romanini et al. (1991)Cancer Res., 51: 789-793; Keyomarsi et al. (1988) J. Biol. Chem., 263:14402-14409). LV stabilizes the ternary complex that inhibitsthymidylate synthase (TS), an enzyme necessary for DNA synthesis.(Longley et al. (2003) Nat. Rev. Cancer, 3(5):330-8). By adding LV to5-FU the overall response rates increased to over 20%. (Id.).

A reduced folate, fotrexorin calcium (CoFactor®)((dl)-5,10,-methylenepteroyl-monoglutamate calcium salt, or[6R,S]-5,10-methylene-THF Ca salt), also known as racemic methyleneTHF,has been suggested as an alternative to LV based on the assumption thatdirect administration of the reduced folate methyleneTHF in place of LVmight offer significant advantages with respect to clinical activity.CoFactor® is a 1:1 mixture of the two diastereoisomers (Odin, E.,Carlsson, G., Frosing, R., Gustaysson, B., Spears, C. P., Larsson, P.A., 1998. Chemical stability and human plasma pharmacokinetics ofreduced folates. Cancer Invest. 16, 447-455). As the [6R]-isomer is thedirectly active co-substrate of TS, it was anticipated that theadministration of CoFactor®, instead of leucovorin, would beadvantageous due to lower inter- and intrapatient variability regardingboth clinical safety and efficacy.

Indeed, in a Phase II Trial in previously untreated metastaticcolorectal cancer, the response rate for CoFactor® was found to be 35%(Saif, M. W, Merritt, J, Robbins J, Stewart J., Schupp, J, 2006. PhaseIII Multicenter Randomized Clinical Trial to Evaluate the Safety andEfficacy of CoFactor®/5-Fluorouracil/Bevacizumab VersusLeucovorin/5-Fluorouracil/Bevacizumab as Initial Treatment forMetastatic Colorectal Carcinoma Clinical Colorectal Cancer, Vol. 6, No.3, 229-234, 2006), and in another phase I/II clinical trial it wasdemonstrated that CoFactor® combined with 5-FU showed clinical benefitin pancreas cancer, defined as stable disease or tumor response, in 40%of patients (Saif, M. W., Makrilia N., Syrigos K., 2010. CoFactor:Folate Requirement for Optimization of 5-Fluouracil Activity inAnticancer Chemotherapy. Journal of Oncology Vol. 1-5). However, apartfrom presenting an unnecessary hepatic detoxification burden, theunnatural (6S)-isomer is a partial competitive inhibitor of the natural[6R]-isomer regarding its effect as co-substrate for TS (Leary, R. P.,Gaumont, Y., Kisliuk, R. L., 1974. Effects of the diastereoisomers ofmethylenetetrahydrofolate on the reaction catalyzed by thymidylatesynthetase. Biochem. Biophys. Res. Commun. 56, 484-488). Furthermore, ina Phase IIb study CoFactor® in colorectal cancer was not demonstrated tobe more efficacious than leucovorin as no significant differencesbetween the study arms with regard to either efficacy or safety could befound, and a planned Phase III study colorectal cancer was discontinuedbefore completion (Press release: ADVENTRX Provides Update on CofactorProgram. Nov. 2, 2007).

There remains a great need for compositions and methods for stabilizingthe ternary complex and enhancing the inhibition of TS. The inventorshave surprisingly discovered that administration of 6R-MTHF increasesplasma levels of 2′-deoxyuridine (dUrd) compared to the administrationof equimolar concentrations of LV. Surprisingly, the inventors havediscovered that administration of 6R-MTHF increases the inhibition of TScompared to the administration of equimolar concentrations of LV.

SUMMARY OF THE INVENTION

The inventors have discovered that equimolar doses of 6R-MTHFsurprisingly produces significantly higher levels of dUrd in bloodplasma compared to with LV when co-administered with 5-FU. The elevationof plasma 2′-deoxyuridine (dUrd) is a marker of TS inhibition. (Ford etal (2002) Clinical Cancer Research, 8(1): 103-109). The presentinvention surprisingly provides methods for increasing plasma dUrdlevels comprising the administration of 6R-MTHF. The methods ofincreasing plasma dUrd increase levels compared to equimolarconcentrations of LV. The present invention also provides methods forincreasing TS inhibition comprising the administration of 6R-MTHF.

Plasma dUrd elevation as a surrogate marker of TS-inhibition hasprogressively become the biomarker of choice. The analysis is alsorelatively simple to perform, by blood sampling and LC-MS/MS analysis.Treatment with TS inhibitors, like 5-FU, causes a rise in intracellularpools of the TS substrate dUMP, which is reflected in raised levels ofthe corresponding nucleoside dUrd, which is largely extracellular andcan be measured in the plasma. Furthermore, elevations of plasma dUrdhave been shown to follow administration of TS inhibitors, and is thus asurrogate marker of TS inhibition (Ford et al (2002)).

The elevation of plasma dUrd levels is understood by the inventors todirectly mirror the current integrated tumor TS-inhibitory state in thebody during 5-FU treatment, reflecting all existing tumor sitesincluding metastases.

The above is strongly supported by the findings that the basic activityof TS in tumors is much higher than in mucosa and that both binding ofthe metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) to TS andTS-inhibitory effect during 5-FU treatment is very much higher in tumorsthan in mucosa and other cells of the body. It is notable that nearly nobinding of FdUMP is observed in tumors without the addition of exogenousfolate (MTHF)(Peters et al. (1991) Eur J Cancer, 27(3): 263-267).

Thus, the plasma dUrd level as a surrogate marker for TS inhibitionclearly stands out against older assays based on e.g. ³H-FdUMPTS-binding, which are only confined to in vitro studies of e.g. biopsiesof tumors or mucosa, or of isolated TS-enzyme. These radiolabel-basedbinding assays are today rarely if ever used and are strongly associatedwith very large degrees of variation (Peters et al. (1991) Eur J Cancer,27(3): 263-267) as well as difficulties to get hold of necessarymaterials for the assays.

It is believed by the inventors that a degree of TS inhibition above 90%is necessary for the tumors to enter into the apoptosis state which isrequired for tumor necrosis to take place. It is further believed that6R-MTHF has the ability to stabilize the above inhibitory ternarycomplex up to the level required for obtaining a TS inhibition of the90% level and above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Increased TS inhibition following administration of 5-FU with 30and 60 mg/m² 6R-MTHF (denoted as “6R”) compared to 30 mg/m² l-LV (“LLV”)(denoted as “LLV 30”) compare to 60 mg/m² racemic (d,l-LV or LV).

FIG. 2: Plasma dUrd levels relative to 30 mg/m² LLV (60 mg/m² of d,l-LVor LV or LV 60) following administration of 5-FU with 30 and 60 mg/m²6R-MTHF (denoted as “6R”).

FIG. 3: Equimolar comparison of LV and 6R-MTHF shown as incrementalplasma dUrd levels at 24 hours after bolus injection of 5-FU 500 mg/m²administered together with bolus injection of 30 mg/m² of 1-LV (denotedas “LLV30”) (60 mg/m² of d,l-LV or LV) or 30 mg/m² of 6R-MTHF (denotedas “M30”). The increments have been calculated as the individualdifferences between dUrd plasma concentrations at 24 hours (t₂₄) minusplasma dUrd concentrations immediately before injection (t₀) for LLVcycles (n=48) and 6R-MTHF cycles (n=18). LLV is the active naturalisomer of LV which is a 50:50 mixture of LLV and the unnatural, not(significantly) active d-LV. The molecular weights of 6R-MTHF and LLVare sufficiently similar as basis for an equimolar comparison. Thedifference between the group has been tested with the Mann-Whitney Utest (p<0.05).

FIG. 4: 6R-MTHF dose dependent increase of incremental plasma dUrdlevels at 24 hours after bolus injection of 5-FU 500 mg/m² administeredtogether with bolus injection of 30 mg/m² LLV (denoted as “LLV30”) (60mg/m² of d,l-LV or LV) or 30 mg/m² (denoted as “M30”) or 60 mg/m² of6R-MTHF (denoted as “M60”). The increments have been calculated as theindividual differences between dUrd plasma concentrations at 24 hours(t₂₄) minus plasma dUrd concentrations immediately before injection (to)for LLV cycles (30 mg/m² n=48) and 6R-MTHF cycles (30 mg/m² n=18; 60mg/m² n=16). The differences between the groups were significant andhave been tested with the the Friedman two-way analysis of variance(p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, [6R]-5,10-methylenetetrahydrofolic acid (6R-MTHF) ora pharmaceutically acceptable salt thereof is employed as a solid formwhich is soluble in water or as a lyophilisate, optionally stabilized byone or more suitable excipients and/or antioxidants such as citric acidor ascorbic acid or salt forms thereof. In one embodiment 6R-MTHF isadministered as one or more IV boluses, each bolus containing 5-1000mg/m² BSA (body surface area), such as 5 mg/m² BSA, such as 7 mg/m² BSA,such as 10 mg/m² BSA, such as 15 mg/m² BSA, such as 30 mg/m² BSA, suchas 60 mg/m² BSA, such as 120 mg/m² BSA, such as 240 mg/m² BSA, such as480 mg/m² BSA, such as 720 mg/m² BSA or such as 960 mg/m² BSA. As usedherein, “bolus” means a method of intravenous administration wherein asingle dose of a pharmaceutical composition is given all at once, unlikeintravenous infusion wherein a single dose is given over at constantconcentration over a period of time.

The dosage depends on the form of therapy, on the form of application ofthe preparation, and on the age, weight, nutrition and condition of thepatient. Treatment can commence with a smaller amount, below the optimumamount, which can be increased in order to achieve the optimum effect.The preferred dose used in therapy ranges between 10 mg and 3,000 mg perday, particularly between 50 mg and 500 mg per day. Administration canbe effected either as a single dose or as a repeated dose.

In one embodiment, 6R-MTHF may be in the form of a free acid, in theform of pharmaceutically acceptable salts, particularly acidic salts, aswell as alkali or alkaline earth metal salts.

In another embodiment, MTHF comprises both diastereomeric isomers,particularly diastereoisomerically pure, natural 6R-MTHF. As usedherein, the term “diastereoisomerically pure” means 6R-MTHF or its saltin isomeric excess over the other isomer greater than about 80%,preferably greater than about 90%, preferably greater than about 95%,more preferably greater than about 97%, even more preferably greaterthan about 99%, more preferably greater than about 99.5% or more, andmost preferably up to 100%, wherein the remainder is the other isomer6S-MTHF.

In another embodiment, the 6R-MTHF is chemically pure. As used herein,the term “chemically pure” means a compound in about 80% chemicalpurity, preferably about 90%, more preferably about 95%, more preferablyabout 97%, more preferably about 98% chemical purity, and mostpreferably 99% or higher than 99%, e.g., 99.5, 99.6, 99.7, 99.8, 99.9 orup to 100% chemical purity, as determined by HPLC. Chemical impuritiesmay include unreacted starting material (including solvents),degradation products of 6R-MTHF (such as tetrahydrofolic acid and itsdegradation products), etc.

Optionally, a pharmaceutical composition comprising 6R-MTHF, forexample, may further comprise at least one anticancer compound. Ananticancer compound may include but is not limited to one or morechemotherapeutic agents, such as but not limited to: nucleic acids, inparticular fluorinated nucleic acids (e.g. 5-flurouracil (5-FU) or ananalog or prodrug thereof), antifolates (e.g. pemetrexed, raltitrexed,lometrexol), topoisomerase inhibitors (e.g. irinotecan, topotecan),antimetabolite drugs (e.g. methotrexate, gemcitabine, tezacitabine),5-FU modulators, alkylating agents (e.g. cyclophosphamide, carmustine),nucleic acid biosynthesis inhibitors (such as mitomycin, anthracyclines(e.g. epirubicin, doxorubicin), platinum derivatives (e.g. cisplatin,oxaliplatin, carboplatin), microtubule disrupting drugs (e.g.paclitaxel, docetaxel, vinolrebine, vincristine), hormone blocking drugs(e.g. tamoxifen), inhibitors of kinases, including but not limited toreceptor and nonreceptor tyrosine kinases (e.g. Iressa, Tarceva, SU5416,PTK787, Gleevec), proteosome inhibitors (e.g. bortezomib), immunemodulators (e.g. levamisole), anti-inflammatory drugs, vascularizationinhibitors, cytokines (e.g. interleukins, tumor necrosis factors), anddrugs that inhibit the activity of cytokines, hormones, or receptors forcytokines or hormones (e.g. the anti-VEGF antibody bevacizumab or“Avastin”). Anticancer compounds may also include monoclonal antibodies,such as but not limited to monoclonal antibodies that bind cytokines,hormones, or hormone receptors (e.g. antibodies that block activation ofEGF or VEGF growth factors, such as Avastin, Erbitux, herceptin), etc.In one embodiment, the 6R-MTHF is administered in combination with atherapeutically effective amount of at least one anticancer compound.When 6R-MTHF is administered in combination with a therapeuticallyeffective amount of at least one anticancer compound a person ofordinary skill in the art would understand that the at least oneanticancer compound can be administered before, after or concurrentlywith 6R-MTHF.

EXAMPLES

The following examples are merely indicative of the nature of thepresent invention, and should not be construed as limiting the scope ofthe invention, nor of the appended claims, in any manner.

Example 1—Treatment with 5-FU and Folates

1. Study Design.

The safety and efficacy of [6R]-5,10-methylenetetrahydrofolate([6R]-MTHF) is analyzed in an open-label, multiple-site, Phase I/II DoseCohort Trial (ISO-CC-005) in patients with stage IV colorectal cancer(mCRC) to determine the safe and tolerable i.v. bolus dose of 6R-MTHF incombination with standard doses of 5-FU (500 mg/m²) alone or incombination with a fixed dose of Bevacizumab, Oxaliplatin or Irinotecanin patients with stage IV colorectal cancer. Among groups of patients(n≥3) ascending 6R-MTHF doses of 30-240 mg/m² BSA are being evaluated inthe presence of various permutations of the cytotoxics, 5-FU alone or5-FU plus oxaliplatin, bevacizumab or irinotecan given at standard doselevels. The study is expected to be completed by year end 2017. Asummary of the study design and each study arm is depicted below inTable 1.

TABLE 1 Initial Doses of the Chemotherapy Agents (Bevacizumab,Oxaliplatin, Irinotecan, and/or 5-FU) and of the Study Drug (6R-MTHF)5-FU Bevacizumab Oxaliplatin^(¶) Irinotecan^(#) At approx. At approx. Atapprox. At approx. 6R-MTHF 35 minutes −180 minutes −60 minutes −60minutes 5-FU^(§)* At approx. 30 (46-hour Treament (infusion 30 to(infusion 15 to (infusion 30 At 0 minute minutes continuous Arm Cohort*90 min) 120 min) to 90 min) (bolus) (bolus)^(a) infusion)^(a) Arm 1Cohort 1 N/A N/A N/A 500 mg/m² 30 mg/m² N/A Cohort 2 N/A N/A N/A 500mg/m² 60 mg/m² N/A Cohort 8 N/A N/A N/A 500 mg/m² 120 mg/m²  N/A Cohort9 N/A N/A N/A 500 mg/m² 240 mg/m²  N/A Arm 2 Cohort 4 N/A 85 mg/m² N/A500 mg/m² 30 mg/m² N/A Cohort 5 N/A 85 mg/m² N/A 500 mg/m² 60 mg/m² N/AArm 3 Cohort 6 N/A N/A 180 mg/m² 500 mg/m² 30 mg/m² N/A Cohort 7 N/A N/A180 mg/m² 500 mg/m² 60 mg/m² N/A Arm 4 Cohort 12 N/A 85 mg/m² N/A 400mg/m²  60 mg/m² ^(a) 2 400 mg/m² Cohort 13 N/A 85 mg/m² N/A 400 mg/m²120 mg/m² ^(a ) 2 400 mg/m² Cohort 14 N/A 85 mg/m² N/A 400 mg/m² 240mg/m² ^(a ) 2 400 mg/m² Arm 5 Cohort 15 5 mg/kg 85 mg/m² N/A 400 mg/m²SP2D^(a,b) 2 400 mg/m² Abbreviation: N/A: not applicable, SP2D: selectedphase 2 dose. ^(¶)The time-point window for Oxaliplatin administrationwill be expanded to allow infusion times of up to 120 minutes, ifnecessary ^(#)The time-point window for Irinotecan administration willbe expanded to allow infusion times of up to 90 minutes, if necessary.^(§)The administered bolus 5-FU dose should not surpass the maximumrecommended daily dose of 1000 mg, regardless of the body surface area.*Cohort #3, Cohort #10 and Cohort #11, originally included in earlierversions of this clinical study protocol, have been erased. ^(a)InTreatment Arm #4 (Cohorts #12, #13, and #14) and Arm #5 (Cohort #15) thetotal dose of 6R-MTHF will be divided into two (2) i.v. bolus injectionsdispensed approximately 30 and 60 minutes after administration of 5-FUbolus injection (at 0 minute), respectively. The continuous 5-FUinfusion will be paused for administration of the second injection of6R-MTHF. ^(b)The dose level of 6R-MTHF in Treatment Arm #4 (MOFOX)assessed as the dose level with the most favourable profile for thefollowing investigation.

In Gothenburg, for almost two decades, clinical, treatment and outcomesdata on all patients with CRC have been collected. Plasma and tissuesamples have been stored in a biobank under appropriate physicalconditions for long term storage. The database and the biobank operateunder the auspices of the relevant ethical and regulatory permissions.Patients having been treated with the standard 5-FU dose, 500 mg/m² plusi.v. bolus LV, 60 mg/m² (equivalent to 30 mg/m² of LLV) were randomlydrawn from the databank.

For all patients, stored plasma samples were used for determination ofdUrd.

The present study is a historical group comparison study. All patientshave been treated with a standard dose of 500 mg 5-FU given as a bolusinjection plus the respective folate 6R-MTHF or LV also given as bolusinjections.

2. Patients Treated with 6R-MTHF.

All patients have been measured during two consecutive treatment cycleswith 5-FU. Values for dUrd were measured immediately before injection of5-FU (to) and after 24 hours (t₂₄). Mean values and standard deviationsfor differences between t₂₄ and to were calculated for the patients oneach dose level of 30 and 60 mg/m² 6R-MTHF, respectively. Some valueshave also been assayed and calculated for 240 mg/m²6R-MTHF.

3. Patients Treated with LV.

Twenty-four patients with metastatic colorectal cancer (mCRC) treatedwith 5-FU plus 60 mg/m² LV were drawn at random from the data base andlevels of dUrd were determined at to and t₂₄ from two treatment cyclesfor each patient and the mean values and standard deviations for thedifferences between t₂₄ and to were calculated in the same way as forthe 6R-MTHF patients. Since LV is a 50:50 mixture of the natural(1-formyl-tetrahydrofolate) and unnatural (d-formyl-tetrahydrofolate)isomers the active isomer constitutes one half of the LV doses given.The molecular weights for LV and 6R-MTHF are very similar and therefore60 mg of LV may be considered as equimolar with 30 mg of 6R-MTHF.

4. Statistical Methods.

The differences between all four groups were tested by means of theFriedman two-way analysis of variance and thereafter the differencebetween the two equimolar groups LV 60 mg/m² and 6R-MTHF 30 mg/m² wastested by means of the Mann-Whitney U test. P-values less than 0.05 wereconsidered significant.

3. Determination of Plasma dUrd.

Plasma dUrd was determined by a method comprising liquid chromatographyfollowed by tandem mass spectrometry broadly summarized as follows.Plasma samples were removed from −80° C. freezer, trichloroacetic acidwas added to the plasma, and the samples mixed and centrifuged. Thesupernatant was filtered in a 10 kDa molecular weight cut-off membranefilter and again centrifuged for 30 min. The solution at the bottom ofthe tube was then ready for LC-MS/MS analysis. Calibration samples wereprepared in the same way using blank plasma samples and differentinternal standard concentrations. The injection volume into LC-MS/MS was40 μl. Deoxyuridine and chlorodeoxyuridine were ionized by electrospraynegative mode. MS parameters were optimized for maximum response of allfolates. A MS/MS acquisition method (multiple reaction monitoring) wasutilized.

Example 2—TS Inhibition

The differences between the first three groups were significant (p=0.04)and also the difference between the two equimolar groups LV 60 mg/m² and[6R]-MTHF 30 mg/m² (p=0.03). An equimolar dose of [6R]-MTHF togetherwith 5-FU gives a significantly higher level of dUrd than does LV. Also,there seems to be a dose-response relationship between increasing[6R]-MTHF doses and increasing levels of TS inhibition as reflected bythe increasing levels of plasma dUrd (See Table 2 and FIG. 1).

TABLE 2 Incremental dUrd at 24 hours after bolus injection of 5-FU plusLLV or 6R- MTHF Mean (t₂₄-t₀) Dose plasma p < Active compound doseadministered No. of dUrd SD LV vs. All mg/m2 BSA mg/m2 BSA cyclespmol/ml pmol/ml 6R Groups¹ LLV 30 60 (d, I-LV) 48 48.7 25.8 0.03 0.046R-MTHF 30 30 18 74.7 52.7 6R-MTHF 60 60 16 91.8 67.6 6R-MTHF 240 240 499.9 ND ¹Except for the 240 mg/m² group.

This study demonstrates that biomodulation of 5-FU with 6R-MTHF ratherthan LV results in higher plasma dUrd and increased TS inhibition. Thisobservation is further supported by the dose dependent inhibition of TSafter increasing doses of 6R-MTHF.

The LV bolus dose of 60 mg/m² is the standard dose used in the so calledNordic treatment regime used widely in Scandinavia. Clinical results aresimilar to those obtained with other regimes when LV is administered byinfusion, often 400 mg over two hours. (Gustaysson et al., (2015)Clinical Colorectal Cancer, 14: 1-10). It is interesting to note themuch higher TS inhibition after 6R-MTHF (FIG. 2).

What is claimed is:
 1. A method of increasing plasma concentration of2′-deoxyuridine (dUrd) in a human subject comprising: a. a step ofadministering 500 mg/m² 5-fluorouracil (5-FU) intravenously as a bolusinjection to said human subject; b. following (a), a step ofadministering 30, 60, 120, or 240 mg/m² [6R]-5, 10-methylenetetrahydrofolate (6R-MTHF) intravenously as one or more bolus injectionsto said human subject; and c. optionally administering a therapeuticallyeffective amount of at least one anticancer agent selected from thegroup consisting of oxaliplatin, irinotecan, and bevacizumab; whereinsaid administration produces an increased plasma concentration of dUrdrelative to dUrd plasma concentration in a human subject followingadministration of an equimolar dose of 5-formyl tetrahydrofolate (LV).2. The method of claim 1, wherein said human subject suffers fromcancer.
 3. The method of claim 2, wherein said cancer is colorectalcancer.
 4. A method of inhibiting thymidylate synthase (TS) in a humansubject comprising: a. a step of administering 500 mg/m² 5-FUintravenously as a bolus injection to said human subject; b. following(a), a step of administering 30, 60, 120, or 240 mg/m² 6R-MTHFintravenously as one or more bolus injections to said human subject; andc. optionally administering a therapeutically effective amount of atleast one anticancer agent selected from the group consisting ofoxaliplatin, irinotecan, and bevacizumab; wherein said administrationproduces an increased inhibition of TS relative to TS inhibition in ahuman subject following administration of an equimolar dose of LV. 5.The method of claim 4, wherein said human subject suffers from cancer.6. The method of claim 5, wherein said cancer is colorectal cancer. 7.The method of claim 1, wherein the 6R-MTHF is administered intravenouslyas more than one bolus injection to said human subject.
 8. The method ofclaim 1, wherein the 6R-MTHF is administered intravenously as two bolusinjections to said human subject.
 9. The method of claim 1, wherein the6R-MTHF is administered intravenously as a bolus injection to said humansubject approximately 30 minutes following step (a).
 10. The method ofclaim 1, where the 6R-MTHF is administered intravenously as a bolusinjection to said human subject approximately 30 and 60 minutesfollowing step (a).
 11. The method of claim 4, where in the 6R-MTHF isadministered intravenously as more than one bolus injection to saidhuman subject.
 12. The method of claim 4, wherein the 6R-MTHF isadministered intravenously as two bolus injections to said humansubject.
 13. The method of claim 4 wherein the 6R-MTHF is administeredintravenously as a bolus injection to said human subject approximately30 minutes following step (a).
 14. The method of claim 4, wherein the6R-MTHF is administered intravenously as a bolus injection to said humansubject approximately 30 and 60 minutes following step (a).
 15. Themethod of claim 1, wherein the 6R-MTHF has a chemical purity greaterthan 90% and in 95% isomeric excess over [6S]-5, 10-methylenetetrahydrofolate (6S-MTHF).
 16. The method of claim 4, wherein the6R-MTHF has a chemical purity greater than 90% and in 95% isomericexcess over [6S]-5, 10-methylene tetrahydrofolate (6S-MTHF).